In affinity assays, a known quantity of a labeled probe competes with or binds to an unknown quantity of unlabeled analyte at binding sites on a target molecule for which the analyte has an affinity. The labeled probe that is bound to the target molecule presents a different measurable phenomenon than the labeled probe that is unbound. Calibration curves relate the presence or quantity of the analyte to the relative amount of bound to unbound labeled probe. The calibration curves are generated by measuring the relative amounts of bound and unbound labeled probe in the presence of known quantities of analyte. In sandwich binding assays, the probe binds to the analyte that is bound to the target molecule. In immunoassays, the analyte is an antigen and the target molecule is an antibody.
In some approaches, the target molecule is affixed to a substrate with properties that causes the emissions from the label to be distinguishable from emissions from a label that is not bound to the target so that the label is displaced farther from the substrate.
During the past decade, there has been a growing interest in plasmonics and in the near-field interactions of fluorophores with metallic structures. Metallic surfaces and particles display surface plasmons, which can result in enhanced and selective excitation of nearby fluorophores. A plasmon is an oscillation of free electron density in a metal particle which can form waves on metal surfaces with the same electric fields and frequencies but shorter wavelengths than electromagnetic waves. In addition, these nearby excited state fluorophores can interact with the photonic mode density (PMD) created by the plasmons, which increases the emission rates and decreases the lifetimes. The PMD is also referred to as the density of states (DoS). The spatial distribution of light from the fluorophore can be changed from the usual omnidirectional distribution to a more narrow spatial distribution, which is determined by wave vector matching at the metallic surfaces.
The use of metals with fluorescence does have some disadvantages. For metal-enhanced fluorescence (MEF), the metal must display a plasmon resonance at wavelengths where its intrinsic absorption is low. This limits the practical metals to Ag, Au, and Al, with a few other metals in occasional use for MEF. There is an optimal distance for metal enhancement near 10 nm from the metal surface because fluorophores at closer distances are often quenched. Metals are lossy and quickly dissipate the optical energy. As a result, MEF often occurs with an increased excitation—relaxation cycling rate.